Thermo Fisher Scientific Imject cBSA Immune Modulator User guide

Brand: Thermo Fisher Scientific

Model: Imject cBSA Immune Modulator

Type: User guide

INSTRUCTIONS

Pierce Biotechnology

PO Box 117

(815) 968-0747

www.thermoscientific.com/pierce

3747 N. Meridian Road

Rockford, lL 61105 USA

(815) 968-7316 fax

Number Description

77150 Imject cBSA Immune Modulator (in PBS), 10mg, supplied lyophilized; when reconstituted with

1mL of ultrapure water, buffer contains 0.1M sodium phosphate, 0.15M NaCl; pH 7.2 with proprietary

stabilizer

77165 Imject cBSA Immune Modulator (in MES buffer), 2mg, supplied lyophilized; when reconstituted

with 0.2mL of ultrapure water, buffer contains 0.1M MES (2-[N-morpholino]-ethanesulfonic acid),

0.15M NaCl; pH 4.7 with proprietary stabilizer

Storage: Upon receipt store product at 4°C. Product is shipped at ambient temperature.

Introduction

Thermo Scientific Imject Cationized BSA (cBSA Immune Modulator) is bovine serum albumin that has been modified by

substituting anionic carboxyl groups with cationic aminoethyl-amide groups. When used as an immunogen, this cationized

BSA (cBSA) stimulates a much higher antibody response than the native form of BSA. In vivo, antibody response is not only

increased but remains elevated for an extended time period. In vitro, much less cBSA than native BSA is required to produce

the same degree of T cell proliferation. The underlying mechanism is related to the form of immunogen that is recognized by

the cells regulating the response. Because of its net positive charge (pI >11), cBSA has a greater affinity for the negatively

charged cell surface membrane of the antigen-presenting cell. Rather than pinocytosis, internalization of cBSA occurs by

receptor-mediated endocytosis, an adsorptive mechanism that results in more efficient uptake and processing of the

immunogen.

Imject Immune Modulator is effective with both haptens and proteins for eliciting an enhanced immunological response

toward the coupled molecule, which eliminates the need to develop protocols for each antigen system. This carrier will

produce an anti-peptide response greater than a traditional carrier and is effective for enhancing the antibody response to

proteins with low isoelectric points.

Important Product Information

• These protocols are designed to yield effective immunogens for a variety of haptens, but are not necessarily optimal for a

specific hapten. Differences in size and structure of haptens will affect conjugation efficiencies. Using a molar excess of

hapten over the carrier protein ensures efficient conjugation. Generally, reacting equal mass amounts of hapten and

carrier protein will achieve sufficient molar excess.

• For haptens with limited solubility, DMSO may be used for solubilization. Use ≤ 30% DMSO in the final conjugation

solution or the carrier protein may irreversibly denature. When using DMSO in the conjugation, add DMSO to the buffer

used for desalting to prevent precipitation in the column. (Dialysis is not compatible with DMSO.)

• If a precipitate has formed during conjugation, centrifuge the material, collect the supernatant and save the precipitate.

Use only the supernatant when purifying the conjugate. Combine the purified conjugate to the precipitate.

• PBS may be used for the final conjugate purification. If the conjugate will be frozen, use the Thermo Scientific Imject

Purification Buffer Salts (Product No. 77159) for purification, which will preserve the product during freeze-thaw cycles.

• To purify the antibodies specific to the peptide, prepare an affinity column by immobilizing the peptide through the same

functional group used to prepare the immunogen. The Thermo Scientific SulfoLink Coupling Gel (Product No. 20402)

contains an activated gel support that will couple peptides via sulfhydryl groups. The Thermo Scientific CarboxyLink

Immobilization Kit (Product No. 44899) uses an amine-containing gel support that will couple peptides via carboxyl

groups using EDC. The peptide affinity column can then be used to specifically bind anti-peptide antibodies from serum,

allowing antibodies against the carrier protein to flow through the column. Peptide-specific antibodies can then be eluted

and recovered.

0136.3

77150 77165

Imject® cBSA Immune Modulator

Pierce Biotechnology

PO Box 117

(815) 968-0747

www.thermoscientific.com/pierce

3747 N. Meridian Road

Rockford, lL 61105 USA

(815) 968-7316 fax

Procedure for Maleimide Activation of cBSA and Hapten Conjugation

The Imject Immune Modulator (in PBS) is supplied in a buffer formulated for conjugation using NHS-ester chemistry. For

example, the crosslinker Sulfo-SMCC contains a maleimide group that will react with free sulfhydryls and a succinimidyl

(NHS ester) group that will react with primary amines. By reacting the reagent to the amines on the cBSA first and then to

the peptide containing a terminal cysteine, all peptide molecules will be coupled with the same orientation.

A. Additional Materials Required

• Sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (Product No. 22322). Other cross-

linkers, such as Sulfo-EMCS (Product No. 22307) or Sulfo-GMBS (Product No. 22324) also may be used.

• Imject Maleimide Conjugation Buffer (Product No. 77164), contains 83mM sodium phosphate, 0.1M EDTA,

0.9M NaCl, 0.02% sodium azide, pH 7.2, with a proprietary stabilizer

• Desalting column (e.g., Polyacrylamide Desalting Columns, Product No. 43240) for removing non-reacted cross-linker

and EDTA. Either desalting or dialysis (Thermo Scientific Slide-A-Lyzer Dialysis Cassettes, Product No. 66810) may be

used in the final purification step.

• Sulfhydryl-containing hapten, 10mg

• Imject Purification Buffer Salts (Product No. 77159), when reconstituted, this buffer contains 0.083M sodium phosphate,

0.9M NaCl and a proprietary stabilizer (see note below)

Note: If the conjugate is to be used for injection within one week, use PBS for the final purification step. If the conjugate

will be frozen, use the Purification Buffer Salts for purification, which will preserve the product during freeze-thaw

cycles.

B. Maleimide Activation and Conjugation Procedure

1. Reconstitute Imject Immune Modulator (in PBS) by adding 1mL of ultrapure water to make a 10mg/mL solution.

2. Immediately before use, prepare a ~10mM solution of Sulfo-SMCC in ultrapure water (5mg/mL).

3. Add 1.5mL of Sulfo-SMCC to the carrier protein and incubate for 60 minutes at room temperature or 30 minutes at 37°C

with periodic gentle mixing.

4. Remove excess crosslinker using a desalting column equilibrated with Imject Maleimide Conjugation Buffer.

5. Dissolve up to 10mg of the sulfhydryl-containing peptide in 2.5mL of Conjugation Buffer.

6. Immediately mix the peptide and activated cBSA and react for 2 hours at room temperature.

7. Purify the conjugate by desalting or dialysis to remove EDTA and sodium azide.

Procedure for Hapten-Carrier Conjugation using EDC

Imject Immune Modulator (in MES buffer) is supplied in a buffer formulated for EDC conjugation chemistry. EDC is a

cross-linker that reacts with carboxyl and amine groups to form stable amide bonds. Because carboxyl groups on cBSA have

been modified, conjugations are site-specific to cBSA amino groups and peptide carboxyl groups.

A. Additional Materials Required

• Hapten or protein, 2mg

• EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride), 10mg (Product No. 77149)

• Imject EDC Conjugation Buffer (Product No. 77162), contains 0.1M MES, 0.9M NaCl, 0.02% NaN3; pH 4.7

• Desalting column (e.g., Dextran Desalting Columns, 5K MWCO, Product No. 43230) or dialysis cassettes (Slide-A-

Lyzer® Dialysis Cassettes, Product No. 66382)

• Imject Purification Buffer Salts (Product No. 77159), when reconstituted, this buffer contains 0.083M sodium phosphate,

0.9M NaCl and a proprietary stabilizer (see note below)

Note: If the conjugate is to be used for injection within one week, use PBS for desalting. If the conjugate will be frozen,

use the Purification Buffer Salts for desalting, which will preserve the product during freeze-thaw cycles.

Pierce Biotechnology

PO Box 117

(815) 968-0747

www.thermoscientific.com/pierce

3747 N. Meridian Road

Rockford, lL 61105 USA

(815) 968-7316 fax

B. EDC Conjugation Procedure

1. Reconstitute Imject Immune Modulator (in MES buffer) by adding 200µL of ultrapure water to make a 10mg/mL

solution.

2. Dissolve up to 2mg of the hapten in 0.5mL of Imject EDC Conjugation Buffer and add it to the cBSA solution.

Note: If the hapten to be coupled is already in a solution free of amines or carboxylic acids and is at pH 4.7-7.2, it may

be added directly to the cBSA solution.

3. Add the peptide/carrier solution to one vial of EDC (10mg) and dissolve by gentle mixing. React for 2 hours at room

temperature.

4. Purify conjugate by desalting or dialysis to remove non-reacted crosslinker and sodium azide.

Additional Information Available on Our Website

Please visit our web site for additional information relating to this product including the following items:

• Tech Tip #18: Block amino groups to prevent polymer formation in peptide-carrier protein conjugations

• Tech Tip #7: Remove air bubbles from columns to restore flow rate

• Tech Tip #29: Degas buffers for use in affinity and gel filtration columns

• Tech Tip #43: Protein stability and storage

Related Thermo Scientific Products

77140 Imject Freund’s Complete Adjuvant, 5 × 10mL

77145 Imject Freund’s Incomplete Adjuvant, 5 × 10mL

77161 Imject Alum, 50mL

77155 Imject Maleimide Activated cBSA, 10mg

77605 Imject Maleimide-Activated mcKLH, 10mg

77661 Imject Maleimide-Activated Blue Carrier Protein, 2mg

General References

Harlow, E. and Lane, D. (1988). Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp. 56-100.

Apple, R.J., et al. (1988). Cationization of protein antigens. IV. Increased antigen uptake by antigen-presenting cells. J Immunol 140:3290-5.

Domen, P.L., et al. (1987). Cationization of protein antigens. III. Abrogation of Oral Tolerance. J Immunol 139: 3195-8.

Muckerheide, A., et al. (1987). Cationization of protein antigens. I. Alteration of immunogenic properties. J Immunol 138:833-7.

Muckerheide, A., et al. (1987). Cationization of protein antigens. II. Alteration of regulatory properties. J Immunol 138:2800-4.

This product (“Product”) is warranted to operate or perform substantially in conformance with published Product specifications in effect at the time of sale,

as set forth in the Product documentation, specifications and/or accompanying package inserts (“Documentation”) and to be free from defects in material and

workmanship. Unless otherwise expressly authorized in writing, Products are supplied for research use only. No claim of suitability for use in applications

regulated by FDA is made. The warranty provided herein is valid only when used by properly trained individuals. Unless otherwise stated in the

Documentation, this warranty is limited to one year from date of shipment when the Product is subjected to normal, proper and intended usage. This

warranty does not extend to anyone other than the original purchaser of the Product (“Buyer”).

No other warranties, express or implied, are granted, including without limitation, implied warranties of merchantability, fitness for any particular

purpose, or non infringement. Buyer’s exclusive remedy for non-conforming Products during the warranty period is limited to replacement of or

refund for the non-conforming Product(s).

There is no obligation to replace Products as the result of (i) accident, disaster or event of force majeure, (ii) misuse, fault or negligence of or by Buyer, (iii)